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Purpose:
To detect nucleic acids from Renibacterium salmoninarum in DNA extracted
from fish tissues or fluids using a nested PCR primer set
Sections:
I. Background
II. References
III. Materials
IV. Procedure
Disclaimer:
The use of trade, firm, or corporation names in this protocol is for the information and
convenience of the reader. Such use does not constitute an official endorsement or
approval by the U.S. Department of Interior or the U.S. Geological Survey of any product
or service to the exclusion of others that may be suitable.
I. Background
The nested PCR (nPCR) test uses two sets of primers that Figure 1. Typical positive and
negative results from nPCR
amplify a segment of the Msa gene that encodes the 57 kDa
major soluble antigen protein. DNA can be extracted from fish
tissues, ovarian fluids or broth culture. The recommended DNA
extraction procedure is outlined in SOP BACT-5 (DNA
extraction for PCR-based detection of Renibacterium
salmoninarum). DNA is subjected to two rounds of PCR
amplification. The first round primer set produces a 383 bp
band while the nested round primer set produces a 320 bp band
(Figure 1). The DNA products from both rounds are visualized *33209 = DNA extracted from a
by agarose gel electrophoresis. culture of R. salmoninarum strain
33209.
Nested PCR is prone to false positives due contamination by first round products.
Laboratories should have four dedicated areas for each stage of the procedure, including:
PCR set-up area: no nucleic acids in this area (PCR reagents only)
DNA template area: area to handle extracted DNA
Amplified template area: area to handle first round template
Gel electrophoresis area: area to handle second round template
Each area should be equipped with dedicated instruments and supplies (e.g. dedicated
pipettors, microcentrifuges, lab jackets, etc.). Work flow should always start in the PCR
set-up area and finish in the gel electrophoresis area (never the opposite direction).
II. References
Chase, D.M. and R. J. Pascho (1998) Development of a nested polymerase chain reaction
for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that
provides a highly sensitive method of detection of the bacterium in salmonid kidney.
Diseases of Aquatic Organisms 34: 223-229.
Pascho, R.J., D. Chase, and C.L. McKibben (1998) Comparison of the membrane-
filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the
polymerase chain reaction to detect Renibacterium salmoninarum in salmonid
ovarian fluid. Journal of Aquatic Animal Health 9: 99-107.
III. Materials
Taq PCR Core Kit: Our laboratory uses the Qiagen Taq PCR Core kit (Qiagen Inc. part
# 201223). Taq polymerase, buffer, dNTPs and loading dyes are also available from a
variety of vendors.
RediloadTM: RediloadTM allows direct loading of PCR reactions into agarose gels.
Purchase from Invitrogen Inc.
Molecular grade water: DNAse- and RNAse-Free water can be purchased from a
variety of vendors. We recommended using a dedicated stock of water for PCR set-up.
Nested PCR Primers: Primers can be synthesized from a variety of vendors. Primers are
typically diluted in molecular grade water to a stock concentration of 100 M (100
pmoles /L). Primers are subsequently diluted to a working concentration of 20 M (20
pmoles /L). Store primers at -20C.
Second Round P4 5-AT TCT TCC ACT TCA ACA GTA CAA GG-3
M38 5-C ATT ATC GTT ACA CCC GAA ACC-3
*New lots of primers should be verified using a known positive sample.
Agarose gel: Molecular grade low EEO agarose can be purchased from various vendors.
100 bp DNA Ladder: DNA ladder for agarose gel electrophoresis can be purchased
from a variety of vendors.
Vendors:
Qiagen Inc.: 1-800-426-8157; www.qiagen.com
Invitrogen Inc.: 1-888.584.8929; www.invitrogen.com
IV. Procedure